1. Field of Invention
The present invention relates to an interferometric detection system and method that can be used, for example, for detection of refractive index changes in picoliter sized samples for chip-scale analyses. The detection system has numerous applications, including universal/RI detection for CE (capillary electrophoresis), CEC (capillary electrochromatography) and FIA, physiometry, cell sorting/detection by scatter, ultra micro calorimetry, flow rate sensing and temperature sensing.
2. Description of the Prior Art
Capillary-based analysis schemes, biochemical analysis, basic research in the biological sciences such as localized pH determinations in tissues and studies in protein folding, detection and study of microorganisms, and the miniaturization of instrumentation down to the size of a chip all require small volume detection. In fact miniaturization of fluid handling systems is at the heart of the genomics and proteomics technology effort. These systems allow one to manipulate single cells or even single macromolecules and it has been recently shown that when liquid handling systems are shrunk to the micron and sub-micron range, small Reynolds numbers and mixing nanoliters in microseconds are possible. Yet, detecting the absolute temperature changes produced in a nanoliter volume T-jump experiment has not been possible. Additionally, the ability to measure biological events such as cold denaturation and binding constants at low temperatures is critically important, but currently limited by existing instrumentation. The potential to perform cellular level investigations and to do high throughput analysis can potentially be realized by using a new generation of analytical instruments based on xe2x80x9cchipsxe2x80x9d, known as miniaturized total analysis systems (xcexc-TAS). In fact, commercial xe2x80x9claboratory on a chipxe2x80x9d devices are now available. It has long been known that the volumetric constraints imposed on the detection system used in xcexc-TAS will dictate the utility of these techniques that are based on microfabrication. Typical injection volumes for xcexc-TAS are in the nanoliter (10xe2x88x929L) to picoliter (10xe2x88x9212L) range and ultimately impart severe constraints on the detection system. In short, the detection volume must be comparable to the injection volume while not sacrificing sensitivity. Yet, the development of xcexc-TAS systems has been accompanied by the implementation, and to a much lesser extent, the improvement of xe2x80x9cconventionalxe2x80x9d detection systems.
Most approaches for xcexc-TAS or on-chip detection have been based on xe2x80x9cconventionalxe2x80x9d optical measurements, primarily absorption, fluorescence or electrochemical. Unfortunately, absorbance measurements are limited in chip-scale techniques because of their inherent path length sensitivity and solute absorbtivity. The fact that the channel dimensions are normally 10-20 xcexcm deep and 20-50 xcexcm wide further exacerbates the S/N limitation for absorbance determinations ultimately limiting picoliter volume detection limits to the range of 0.1-0.01 mM.
With the advent of lasers, light sources possessing unique properties including high spatial coherence, monochromaticity and high photon flux, unparalleled sensitivity and selectivity in chemical analysis is possible. The advantages of using lasers in micro-chemical analysis are well known and have been demonstrated thoroughly. Over the past five years, technical advances in the laser have lead to reduced cost, enhanced reliability and availability of new wavelengths or multi-wavelength scanning systems. The result has been the demonstration of a number of high sensitivity/micro-volume detection methodologies for universal analysis. For example laser-induced fluorescence (LIF) can provide extremely low detection limits, with most laboratories able to detect as few as 105 molecules. In fact, recent developments in ultra-high sensitivity LIF have allowed single molecule detection to be performed xe2x80x98on-chipxe2x80x99. While fluorescence is an inherently sensitive detection method, it can be expensive to implement and is only applicable to solutes that are either, naturally fluorescent (the number of such molecules is actually quite small) or that can be chemically modified to fluoresce. Other approaches to on-chip detection have primarily included thermal conductivity, electroluminescence and electrochemical methods. However, these technologies are also expensive and hard to implement.
Refractive index detection is still a common technique used in chemical and biochemical analysis that has been successfully applied to several small volume analytical separation schemes. For various reasons, RI detection represents an attractive alternative to fluorescence and absorbance. First, RI detection is relatively simple. Second, it can be used with a wide range of buffer systems. Finally, RI detection is universal, theoretically allowing detection of any solute, making it particularly applicable to solutes with poor absorption or fluorescence properties. However, for a number of reasons, attempts toward implementation of RI detection in chip-scale analyses has been somewhat problematic.
Previous attempts for on-chip RI detection have generally involved the use of either waveguiding or interferometry. Among these techniques are the Mach-Zender approach, the porous silicon-based optical interferometer, surface plasmon resonance (SPR) (and related) techniques, the xe2x80x98on-chipxe2x80x99 spiral-shaped waveguide refractometer, and the holographic forward scatter interferometer. While each of the aforementioned RI measurement techniques can produce impressive results, they are all limited when applied to on-chip detection with chip scale analyses. In general, the path length dependency of evanescent wave-based techniques like SPR or the Mach Zender interferometer, demands a long sensing region be in contact with the separation fluid resulting in an optical xe2x80x9cdetectionxe2x80x9d volume too large to be compatible with chip-scale analyses.
The porous silicon-based optical interferometer (a Fabry-Perot system) can provide pico- and even femtomolar analyte sensitivity, but for the RI signal to be produced, this sensor requires (as do the SPR sensors) that the exogenous xe2x80x98reporterxe2x80x99 molecules be attached to the surface of the silicon and subsequently bind to the desired or target solute. This methodology of using molecular recognition which leads to an RI change can be used as an on-chip detector, provides solute selectively, leading inherently to high sensitivity, but is limited by reaction kinetics and the need to do sophisticated biochemistry and surface immobilization. These chemistries are normally diffusion limited and thus take time. In addition, solute events produced in CE, FIA or chip scale HPLC must be detected as they traverse the detector. Temporal constraints can be severe and range from 10""s of milli-seconds to several minutes. Thus the peak must be sensed or analyzed in the probe volume during the elution time. Furthermore, technologies such as SPR do not provide the option to directly monitor xcexc-Vol. temperature changes as are needed to study, for example, reaction kinetics or to perform on-chip flow rate sensing.
The holographic forward scatter interferometer is thus far, the most promising approach for on-chip universal or RI detection in CE, and uses a holographic grating and a forward scattering optical configuration. However, while research on this technique has clearly shown the potential for doing on-chip RI sensing, the sensitivity of the forward scatter technique employed is inherently limited because it is has a single pass optical configuration, e.g. the probe beam traverses only once through the detection channel.
In view of the foregoing, a need still remains for an RI detection technique that is sensitive, universal can probe ultra-small volumes, is compatible with the chip-based format and can be employed for temperature and flow rate sensing of ultra-small volumes.
The present invention fulfills the need for a new sensing methodology applicable to xcexc-TAS through provision of an interferometric detection system and method that circumvent the drawbacks of xe2x80x98standardxe2x80x99 interferometric methods and the limitations of the forward scatter technique. The system includes a source of coherent light, such as a diode or Hexe2x80x94Ne laser, a channel of capillary dimensions that is preferably etched in a substrate for reception of a sample to be analyzed, and a photodetector for detecting backscattered light from the sample at a detection zone.
The laser source generates an easy to align simple optical train comprised of an unfocused laser beam that is incident on the etched channel for generating the backscattered light. The backscattered light comprises interference fringe patterns that result from the reflective and refractive interaction of the incident laser beam with the channel walls and the sample. These fringe patterns include a plurality of light bands whose positions shift as the refractive index of the sample is varied, either through compositional changes or through temperature changes, for example. The photodetector detects the backscattered light and converts it into intensity signals that vary as the positions of the light bands in the fringe patterns shift, and can thus be employed to determine the refractive index (RI), or an RI related characteristic property, of the sample. A signal analyzer, such as a computer or an electrical circuit, is employed for this purpose to analyze the photodetector signals, and determine the characteristic property of the sample.
Preferably, the channel has a generally hemispherical cross sectional shape. A unique multi-pass optical configuration is inherently created by the channel characteristics, and is based on the interaction of the unfocused laser beam and the curved surface of the channel, that allows interferometric measurements in small volumes at high sensitivity. Additionally, if a laser diode is employed as the source, not only does this enable use of wavelength modulation for significant improvements in signal-to-noise ratio, but it also makes it possible to integrate the entire detector device directly onto a single microchip.
The detector can be employed for any application that requires interferometric measurements, however, the detector is particularly attractive for making universal solute quantification, temperature and flow rate measurements. In these applications, the detector provides ultra-high sensitivity due to the multi-pass optical configuration of the channel. In the temperature measuring embodiment, the signal analyzer receives the signals generated by the photodetector and analyzes them using the principle that the refractive index of the sample varies proportionally to its temperature. In this manner, the signal analyzer can calculate temperature changes in the sample from positional shifts in the detected interference fringe patterns.
In the flow measuring embodiment, the same principle is also employed by the signal analyzer to identify a point in time at which a thermal perturbation is detected in a flow stream in the channel. First, a flow stream whose flow rate is to be determined, is locally heated at a point that is known distance along the channel from the detection zone. The signal analyzer for this embodiment includes a timing means or circuit that notes the time at which the flow stream heating occurs. Then, the signal analyzer determines from the positional shifts of the light bands in the interference fringe patterns, the time at which the thermal perturbation in the flow stream arrives at the detection zone. The signal analyzer then determines the flow rate from the time interval and distance values.